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Mmessage machine t7 ultra kit

Manufactured by Thermo Fisher Scientific
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The MMessage Machine T7 Ultra Kit is a lab equipment product designed for in vitro transcription. It enables the synthesis of capped and polyadenylated messenger RNA (mRNA) from DNA templates containing a T7 promoter.

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Lab products found in correlation

Rabbit reticulocyte lysate, by Promega (2 mentions) Bamhi, by New England Biolabs (2 mentions) Dual glo luciferase assay, by Promega (2 mentions) Dynabead, by Thermo Fisher Scientific (2 mentions) Rneasy plus mini kit, by Qiagen (2 mentions) Wild type human cx26 hcx26 cdna, by OriGene (2 mentions) Pgem ha vector, by Promega (2 mentions) Quickchange 2 site directed mutagenesis kit, by Agilent Technologies (2 mentions) Peasy t vector, by Promega (1 mentions) Rneasy kit, by Qiagen (1 mentions)

6 protocols using mmessage machine t7 ultra kit

1

Cloning and Injecting ZFPM2 mRNA

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The human wild-type and mutant ZFPM2 sequences were cloned into the pEasy-T vector (Promega, USA). The capped and poly(A) tailed mRNA of hZFPM2 and its mutant were synthesized in vitro by transcribing with T7 RNA polymerase using the mMessageMachine T7 Ultra Kit (Ambion, Cat #AM1345, USA). The embryos of the transgenic cmlc2 were as follows: eGFP was collected for the microinjection at the single-cell stage. The experimental groups included the Mut, WT and control groups. For the control group, an equal volume of solution was injected. One hundred embryos were collected from each injection group and the control group. The purified mRNA of 450 pg was injected into the embryos at the single-cell stage. A minimum of three independent experiments was performed for the wild-type and mutant ZFPM2 mRNA injections.
Qian Y., Xiao D., Guo X., Chen H., Hao L., Ma X., Huang G., Ma D, & Wang H. (2017). Multiple gene variations contributed to congenital heart disease via GATA family transcriptional regulation. Journal of Translational Medicine, 15, 69.
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2

Dicistronic Reporter Construct Assay

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The dicistronic reporter construct, containing the Renilla luciferase sequence after the 5′ UTR followed by the CrPV IRES and the firefly luciferase sequence, has been previously described42 (link). The reporter construct plasmid was linearized with BamHI (New England Biolabs) and transcribed in vitro with an ARCA cap and poly (A)-tailed using the mMessage Machine T7 Ultra Kit (Ambion). The mRNAs were purified with RNeasy Plus Mini Kit (Qiagen) with RNase-free H2O. Human eIF1A and mutants (V55A, K56A and K67A, with anti-Flag antibodies, 0.15mg/ml, 2μl per reaction per reaction) were purified with Dyna beads (Invitrogen) from HEK293 cells with transient transfection of plasmids encoding Flag-tagged eIF1A and eIF1A mutants. In vitro translation reactions (50μl per reaction) were carried out using Rabbit Reticulocyte Lysate (Promega, 35μl per reaction) with 2mM magnesium acetate and 60mM potassium acetate, which was incubated at 30°C for 100 min. Translation of the reporter genes was measured using the Dual-Glo luciferase assay (Promega). Three independent experiments were performed.
Yi T., Arthanari H., Akabayov B., Song H., Papadopoulos E., Qi H.H., Jedrychowski M., Güttler T., Guo C., Luna R.E., Gygi S.P., Huang S.A, & Wagner G. (2015). eIF1A augments Ago2-mediated Dicer-independent miRNA biogenesis and RNA interference. Nature communications, 6, 7194.
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3

Capped and Tailed SNRNP200 mRNA Synthesis

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Capped and tailed mRNAs of SNRNP200 WT (hSnrnp200 WT) and SNRNP200 c.C6088T (hSnrnp200 Arg2030Cys) were achieved from the circular plasmids cut by the endonuclease XmaI AcpxT7- SNRNP200WT and AcpxT7- SNRNP200c.C6088T with the mMESSAGE MACHINE T7 Ultra Kit (Ambion, Austin, TX, USA). RNeasy Kit (Qiagen, Hilden, Germany) was employed to erase extra nucleotides, especially DNA aiming at purifying the synthesized mRNAs.
Zhang T., Bai J., Zhang X., Zheng X., Lu N., Liang Z., Lin L, & Chen Y. (2021). SNRNP200 Mutations Cause Autosomal Dominant Retinitis Pigmentosa. Frontiers in Medicine, 7, 588991.
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4

Dicistronic Reporter Construct Assay

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The dicistronic reporter construct, containing the Renilla luciferase sequence after the 5′ UTR followed by the CrPV IRES and the firefly luciferase sequence, has been previously described42 (link). The reporter construct plasmid was linearized with BamHI (New England Biolabs) and transcribed in vitro with an ARCA cap and poly (A)-tailed using the mMessage Machine T7 Ultra Kit (Ambion). The mRNAs were purified with RNeasy Plus Mini Kit (Qiagen) with RNase-free H2O. Human eIF1A and mutants (V55A, K56A and K67A, with anti-Flag antibodies, 0.15mg/ml, 2μl per reaction per reaction) were purified with Dyna beads (Invitrogen) from HEK293 cells with transient transfection of plasmids encoding Flag-tagged eIF1A and eIF1A mutants. In vitro translation reactions (50μl per reaction) were carried out using Rabbit Reticulocyte Lysate (Promega, 35μl per reaction) with 2mM magnesium acetate and 60mM potassium acetate, which was incubated at 30°C for 100 min. Translation of the reporter genes was measured using the Dual-Glo luciferase assay (Promega). Three independent experiments were performed.
Yi T., Arthanari H., Akabayov B., Song H., Papadopoulos E., Qi H.H., Jedrychowski M., Güttler T., Guo C., Luna R.E., Gygi S.P., Huang S.A, & Wagner G. (2015). eIF1A augments Ago2-mediated Dicer-independent miRNA biogenesis and RNA interference. Nature communications, 6, 7194.
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5

In vitro Characterization of Connexin 26 Mutations

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Wild-type human Cx26 (hCx26) cDNA was purchased from OriGene and was subcloned in the pGEM-HA vector (Promega) for in vitro translation. Mutations of hCx26 were produced with Quick-Change II Site-Directed Mutagenesis kits (Agilent Technologies). DNA sequencing performed at the New Jersey Medical School Molecular Resource Facility confirmed the amino acid substitutions. Nhe1-linearized hCx26 wild type, Cx43 and mutant DNAs were transcribed in vitro to cRNAs using the T7 Ultra mMessage Machine kit (Ambion). Electrophysiological data were collected using the two-electrode voltage-clamp technique as in our previous studies (Lopez et al., 2013a (link)).
García I.E., Maripillán J., Jara O., Ceriani R., Palacios-Muñoz A., Ramachandran J., Olivero P., Pérez-Acle T., González C., Sáez J.C., Contreras J.E, & Martínez A.D. (2015). Keratitis-Ichthyosis-Deafness syndrome-associated Cx26 mutants produce nonfunctional gap junctions but hyperactive hemichannels when co-expressed with wild type Cx43. The Journal of investigative dermatology, 135(5), 1338-1347.
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6

In vitro Characterization of Connexin 26 Mutations

Cited 1 time
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Wild-type human Cx26 (hCx26) cDNA was purchased from OriGene and was subcloned in the pGEM-HA vector (Promega) for in vitro translation. Mutations of hCx26 were produced with Quick-Change II Site-Directed Mutagenesis kits (Agilent Technologies). DNA sequencing performed at the New Jersey Medical School Molecular Resource Facility confirmed the amino acid substitutions. Nhe1-linearized hCx26 wild type, Cx43 and mutant DNAs were transcribed in vitro to cRNAs using the T7 Ultra mMessage Machine kit (Ambion). Electrophysiological data were collected using the two-electrode voltage-clamp technique as in our previous studies (Lopez et al., 2013a (link)).
García I.E., Maripillán J., Jara O., Ceriani R., Palacios-Muñoz A., Ramachandran J., Olivero P., Pérez-Acle T., González C., Sáez J.C., Contreras J.E, & Martínez A.D. (2015). Keratitis-Ichthyosis-Deafness syndrome-associated Cx26 mutants produce nonfunctional gap junctions but hyperactive hemichannels when co-expressed with wild type Cx43. The Journal of investigative dermatology, 135(5), 1338-1347.
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